Methods and compositions related to immunogenic fibrils

ABSTRACT

Embodiments of the invention are directed to fibrillar adjuvants. Epitopes assembled into nanofibers by a short synthetic fibrillization domain elicited high antibody titers in the absence of any adjuvant.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/263,213 filed Nov. 20, 2009, which is incorporated herein byreference in its entirety.

This invention was made with government support under DE017703 andEB009701 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

I. Field of the Invention

Embodiments of this invention are directed generally to biology,medicine, and immunology. Certain aspects are directed to immunogenicfibrils and their use in inducing an immune response.

II. Background

The development of vaccines and other immunotherapies has beenchallenged by imprecise antigen display and the use of heterogeneousimmune adjuvants whose mechanisms of action are complex and incompletelyunderstood. Synthetic peptides are useful as antigens because theirprecise chemical definition allows one to specify the exact epitopesagainst which an immune response is to be raised. However, peptides arepoorly immunogenic by themselves and require co-administration withstrong adjuvants, a process that sacrifices the chemical definition thatpeptides possess initially and complicates their development andregulatory approval Lambrecht et al., 2009; Marrack et al., 2009;Purcell et al., 2007). Although several adjuvants have been investigatedfor peptide immunotherapies to date, current strategies such asparticulates (Marrack et al., 2009; Wendorf et al., 2006), oil emulsions(Daftarian et al., 2006), toll-like receptor ligands (Ishii and Akira,2007), ISCOMs (Maraskovsky et al., 2009), and other biologically sourcedmaterials (McSorley et al., 2002; Sun et al., 2009) utilize chemicallyor structurally heterogeneous materials, making characterization andmechanistic understanding challenging. This situation has motivated thepursuit of self-adjuvanting or adjuvant-free systems (Bettahi et al.,2009; Cao et al., 2008; Toth et al., 2008).

There remains a need for additional immunogenic compositions to induceimmune responses for treating microbial infection and other pathogenicconditions.

SUMMARY OF THE INVENTION

Strong antibody responses have been observed in mice without theco-administration of any additional adjuvant by non-covalentlyassembling a T and B cell epitope peptide into nanofibers using a shortC-terminal peptide extension. The inventors have discovered thatself-assembling peptides are useful as chemically defined adjuvants. Inphysiological conditions, these peptides self-assembled into long,unbranched fibrils that displayed the epitope on their surfaces. IgG1,IgG2a, and IgG3 were raised against epitope-bearing fibrils in levelssimilar to the epitope peptide delivered in complete Freund's adjuvant(CFA), and

IgM production was even greater for the self-assembled epitope. Thisresponse was dependent on self-assembly, and the self-assemblingsequence was not immunogenic by itself, even when delivered in CFA.

Certain embodiments of the invention are directed to immunogeniccompositions comprising a peptide fibril coupled to a plurality ofantigens. In certain aspects, the peptide fibril comprises a pluralityof self-assembling peptides. The peptide fibril can have a length of atleast 0.25, 0.5, 1, 10, 50 to 10, 25, 50, 100 μm, including all valuesand ranges there between. In certain aspects, the peptide fibril has amolecular weight of at least 1,000, 5,000, 10,000, 100,000 da to 1×10⁶,1×10⁷, 7×10⁸ da, including all values and ranges there between. In otheraspects the self-assembling peptides form a beta-sheet rich fibril. Infurther aspects, the self-assembling peptide comprises an amino acidsequence of QQKFQFQFEQQ (SEQ ID NO:1; KFQFQFE (SEQ ID NO:2); QQRFQFQFEQQ(SEQ ID NO:3); QQRFQWQFEQQ (SEQ ID NO:4); FEFEFKFKFEFEFKFK (SEQ IDNO:5); QQRFEWEFEQQ (SEQ ID NO:6); QQXFXWXFQQQ (SEQ ID NO:7) (Where Xdenotes ornithine); FKFEFKFEFKFE (SEQ ID NO:8); FKFQFKFQFKFQ (SEQ IDNO:9); AEAKAEAKAEAKAEAK (SEQ ID NO:10); AEAEAKAKAEAEAKAK (SEQ ID NO:11);AEAEAEAEAKAKAKAK (SEQ ID NO:12); RADARADARADARADA (SEQ ID NO:13);RARADADARARADADA (SEQ ID NO:14); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQID NO:15); EWEXEXEXEX (SEQ ID NO:16) (Where X=V, A ,S, or P); (SEQ IDNO:17) (Where X=V, A, S, or P); KWKVKVKVKVKVKVK (SEQ ID NO:18);LLLLKKKKKKKKLLLL (SEQ ID NO:19; VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO:20;VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO:21; KVKVKVKVKDPPSVKVKVKVK (SEQ IDNO:22; or VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO:23); VKVKVKTKVDPPTKVKTKVKV(SEQ ID NO:24). In certain aspects, the self-assembling peptide is 6, 7,8, 9, 10, 11, 12, 13, 14, 15 to 15, 20, 25, 30, 35, 40 amino acids inlength, including all values and ranges there between. In certainaspects, more than one self-assembling peptide is present in a peptidefibril. In certain aspects the self assembling peptides comprise animmunogenic peptide or an antigen. In certain aspects the antigen(s) arepolypeptides. In a further aspect the polypeptides are covalentlycoupled to the peptide fibril. In still a further aspect thepolypeptides are covalently coupled to the peptide fibril via a cutinasepolypeptide.

In a further aspect, the antigen is covalently coupled to theself-assembling peptide. In a further aspect, the antigen is covalentlycoupled to the N and/or C terminus of the self-assembling peptide. Instill further aspects, the antigen is covalently coupled to the carboxyterminus of the self-assembling peptide. In certain aspects the ratio ofantigen to self-assembling peptide is 1:1000, 1:100: 1:10, or 1:1,including all values and ranges there between. Antigens can be microbialantigens, such as viral, fungal, or bacterial; or therapeutic antigenssuch as antigens associated with cancerous cells or growths, orautoimmune disorders. In certain aspects, the antigens are peptides,lipids, carbohydrates, or other immunogenic molecules. In a furtheraspect, the peptides are 5, 10, 15, 20 to 15, 20, 30, 40 amino acids inlength, including all values and ranges there between. The antigens canbe T cell and/or B cell epitopes.

Further embodiments include self-assembling antigens comprising anantigen coupled to a fibril-forming peptide.

Certain embodiments are directed to methods of inducing an immuneresponse comprising administering an antigenic fibril, comprising aself-assembling peptide coupled to an antigen, in an amount sufficientto induce an immune response.

Further embodiments are directed to methods of treating a subject havingor at risk of developing a microbial infection, cancer, or othercondition that can be treated by inducing an immune response comprisingadministering to the subject an effective amount of a compositiondescribed herein.

As used herein, the term “antigen” is a molecule capable of being boundby an antibody or T-cell receptor. An antigen is additionally capable ofinducing a humoral immune response and/or cellular immune responseleading to the production of B- and/or T-lymphocytes. The structuralaspect of an antigen that gives rise to a biological response isreferred to herein as an “antigenic determinant.” B-lymphocytes respondto foreign antigenic determinants via antibody production, whereasT-lymphocytes are the mediator of cellular immunity. Thus, antigenicdeterminants or epitopes are those parts of an antigen that arerecognized by antibodies, or in the context of an MHC, by T-cellreceptors. An antigenic determinant need not be a contiguous sequence orsegment of protein and may include various sequences that are notimmediately adjacent to one another.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. The embodiments in the Example section are understood to beembodiments of the invention that are applicable to all aspects of theinvention.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation ofthese terms, when used in the claims and/or the specification includesany measurable decrease or complete inhibition to achieve a desiredresult.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Furthermore, compositions and kits of the invention canbe used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” It is also contemplatedthat anything listed using the term “or” may also be specificallyexcluded.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1B Schematic (A) and sequences (B) of epitope-bearingself-assembling peptides. The Q11 domain assembles into fibrillaraggregates, displaying the epitope sequence at the end of a flexiblespacer.

FIGS. 2A-2F Q11-based peptides self-assembled into β-sheet fibrils thatdisplayed functional epitopes on their surfaces. Q11 (A) self-assembledinto long, unbranched fibrils that did not bind streptavidin-gold (B).O-Q11 (C) also formed long, unbranched fibrils, and biotin-O-Q11 boundstreptavidin-gold (D), indicating availability of the N-terminus on thefibril surface. O-Q11 possessed a predominant β-sheet structure by CD(E). OVA antisera reacted similarly to ELISA plates coated with OVA orO-Q11 (F). Each point represents one mouse's serum; bars represent themean. *p<0.01 compared to OVA coat/OVA in PBS injection, by ANOVA withTukey HSD post-hoc testing. n.s., not statistically different.

FIGS. 3A-3D Fibrillization by the Q11 domain strongly adjuvanted IgGresponses to OVA. Similar titers of total IgG were raised against O-Q11delivered in PBS and OVA delivered in CFA, whereas Q11 or OVA deliveredin PBS did not elicit a response (A). Q11 was non-immunogenic even inCFA, whereas CFA increased IgG titers for O-Q11 (B). O-Q11 antisera werestrongly cross-reactive to OVA-coated ELISA plates and showed a smallamount of reactivity to Q11-coated plates that was not statisticallysignificant (C). The adjuvant activity of Q11 was entirely dependent oncovalent conjugation between the fibrillizing domain and epitope domain,as mixtures of Q11 and OVA did not raise any OVA-specific IgG (D). Eachpoint represents one mouse; bars represent the mean. *p<0.01 by ANOVAwith Tukey HSD post-hoc testing, compared with OVA in PBS, or betweengroups as indicated.

FIG. 4 Antibody isotypes in sera from mice immunized with peptides. Eachpoint represents one mouse; bars represent the mean. *p<0.01 comparedwith OVA delivered in PBS by ANOVA with Tukey HSD post-hoc test. §p<0.05as indicated.

FIGS. 5A-5C IFN-γ, IL-2, and IL-4 production in peptide-challenged(triangles) and unchallenged (circles) splenocyte cultures. Each pointrepresents one mouse; bars represent the mean. *p<0.01 compared withcorresponding unchallenged control, by ANOVA with Tukey HSD post-hoctest.

FIG. 6 Shows total IgG ELISA absorbance values for serially dilutedserum from individual mice, corresponding to the titer calculationsshown in FIG. 5. Each line represents one mouse. Mice receiving O-Q11 inPBS, OVA in CFA, Q11, and OVA in PBS are shown. The inset shows anexpanded scale, for visualizing the low absorbance values of the Q11 andnegative control mice.

FIG. 7 Total IgG antibodies by mice immunized withGFP-cutinase-phosphonate-Q11 fibrils, cutinase-GFP emulsified in CFA, orcutinase-GFP in PBS.

DETAILED DESCRIPTION OF THE INVENTION

The development of vaccines and other immunotherapies has beenchallenged by imprecise antigen display and the use of heterogeneousimmune adjuvants whose mechanisms of action are complex and incompletelyunderstood. Synthetic peptides are useful as antigens because theirprecise chemical definition allows one to specify the exact epitopesagainst which an immune response is to be raised. However, peptides arepoorly immunogenic by themselves and require co-administration withstrong adjuvants, a process that sacrifices the chemical definition thatpeptides possess initially and complicates their development andregulatory approval (Lambrecht et al., 2009; Marrack et al., 2009;Purcell et al., 2007). Although several adjuvants have been investigatedfor peptide immunotherapies to date, current strategies such asparticulates (Marrack et al., 2009; Wendorf et al., 2006), oil emulsions(Daftarian et al., 2006), toll-like receptor ligands (Ishii and Akira,2007), ISCOMs (Maraskovsky et al., 2009), and other biologically sourcedmaterials (McSorley et al., 2002; Sun et al., 2009) utilize chemicallyor structurally heterogeneous materials, making characterization andmechanistic understanding challenging. This situation has motivated thepursuit of self-adjuvanting or adjuvant-free systems (Bettahi et al.,2009; Cao et al., 2008; Toth et al., 2008).

A broad goal in the field of biomaterials is to produce syntheticscaffolds capable of presenting multiple cell-interactive components inspatially resolved networks (Lutolf and Hubbell, 2005; Place et al.,2009). To accomplish this, supramolecular self-assembly is rapidlybecoming a synthetic method of choice (Silva et al., 2004; Jung et al.,2009; Collier, 2008). One strategy that has received attention recentlyis based on fibrillized peptides, peptidomimetics, and peptidederivatives, which are being explored for a variety of biomedical andbiotechnological applications, most notably as scaffolds forregenerative medicine (Davis et al., 2005; Tysseling-Mattiace, 2008) anddefined matrices for cell culture (Genove et al., 2005; Horii et al.,2007). In these applications, self-assembled materials provide severaladvantages, including multifunctionality, multivalency, syntheticdefinition, molecular specificity, and control over the nanoscalepositioning of ligands and other biomolecular features (Collier, 2008).Self-assembled, multi-component matrices for cell culture using a shortfibrillizing peptide, Q11 (Ac-QQKFQFQFEQQ-Am) (SEQ ID NO:1) have beendescribed (Jung et al., 2009; Collier, 2008; Collier and Messersmith,2003; Jung et al., 2008). This peptide, like other previously reportedshort fibrillizing peptides (Horii et al., 2007; Holmes et al., 2000;Aggeli et al., 1997; Gras et al., 2008), β-hairpins (Schneider et al.,2002), peptide-amphiphiles (Tysseling-Mattiace, 2008; Hartgerink et al.,2001), and peptide derivatives (Zhou et al., 2009), self-assembles insalt-containing aqueous environments to form networks of β-sheet-richnanofibers. It is also capable of displaying functional amino acidsequences or chemical groups on the surface of its self-assembledfibers. For example, adding cell-binding amino acid sequences to theN-terminus of Q11 leads to self-assembled fibrils that functionallypresent the cell-binding peptides on their surfaces (Jung et al., 2009).Q11 with an N-terminal cysteine and a C-terminal thioester can undergonative chemical ligation after assembly, which can be used to stiffenthe fibrillar network (Jung et al., 2008). These peptides can also bemixed to display precise combinations of different ligands (Jung et al.,2009).

During initial development as scaffolds for regenerative medicine, Q11and other self-assembling peptide-based materials have been found to beminimally immunogenic. In previous work, Q11 and Q11 with N-terminalcell-binding RGDS sequences elicited little to no antibody responses inmice (Jung et al., 2009). Negligible tissue responses were also observedfor n-sheet fibrillizing RAD16-II peptide assemblies injected within rat(Hsieh et al., 2006) or mouse (Davis et al., 2005) myocardium. Lowantibody titers have also been reported in rabbits and goats for RAD16peptides (Holmes et al., 2000). One study observed an inflammatoryresponse to RAD16 peptides in rats, but the causes were not known(Dubois et al., 2008). The minimal immunogenicity of these materialsobserved to date is clearly advantageous for applications inregenerative medicine, but previous work has focused largely on aminoacid sequences that are already found in endogenous proteins, forexample the RGDS cell-binding sequence from fibronectin (Jung et al.,2009; Guler et al., 2006). Because of this, strong epitopes haveeffectively been avoided. In the present work, it was sought todetermine the extent to which the previously observed low immunogenicityalso applied to peptide sequences containing stronger epitopes. Theresults show a surprisingly robust antibody response generated against aself-assembled B and T cell epitope, indicating that self-assembledpeptides can serve as powerful chemically defined adjuvants.

I. Self-Assembling Peptides

Certain aspects of the invention include self-assembling peptides. Asused herein, the term “self-assembling peptide” refers to peptides thatare able to spontaneously associate and form stable structures. In oneembodiment, a self-assembling peptide of the present invention comprisesan amino acid sequence of QQKFQFQFEQQ; KFQFQFE; QQRFQFQFEQQ;QQRFQWQFEQQ; FEFEFKFKFEFEFKFK; QQRFEWEFEQQ; QQXFXWXFQQQ (Where X denotesornithine); FKFEFKFEFKFE; FKFQFKFQFKFQ; AEAKAEAKAEAKAEAK;AEAEAKAKAEAEAKAK; AEAEAEAEAKAKAKAK; RADARADARADARADA; RARADADARARADADA;SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG; EWEXEXEXEX (Where X=V, A, S, or P);(Where X=V, A, S, or P); KWKVKVKVKVKVKVK; LLLLKKKKKKKKLLLL;VKVKVKVKVDPPTKVKVKVKV; VKVKVKVKVDPPTKVKTKVKV; KVKVKVKVKDPPSVKVKVKVK;VKVKVKVKVDPPSKVKVKVKV; VKVKVKTKVDPPTKVKTKVKV or conservatively modifiedvariants thereof. Self-assembling peptides may further comprise othercompounds, for example, immunogenic peptides.

Certain peptides that comprise of alternating hydrophilic andhydrophobic amino acids self-assemble to form an exceedingly stablebeta-sheet macroscopic scaffold (U.S. Pat. Nos. 5,955,343 and 5,670,483,each of which is incorporated herein by reference).

Many self-complementary peptides have identical compositions and length;such as EAK16, KAE16, RAD16, RAE16, and KAD16; have been analyzedpreviously (Table 1).

TABLE 1 Representative Self-Assembling peptides SEQ ID NameSequence (n-->c) NO: Modulus Structure RADA16-I n-RADARADARADARADA-c 29I β RGDA16-I n-RADARGDARADARGDA-c 30 I r.c RADA8-I n-RADARADA-c 31 Ir.c. RAD16-II n-RARADADARARADADA-c 32 II β RAD8-II n-RARADADA-c 33 IIr.c. EAKA16-I n-AEAKAEAKAEAKAEAK-c 34 I β EAKA8-I n-AEAKAEAK-c 35 I r.c.RAEA16-I n-RAEARAEARAEARAEA-c 36 I β RAEA8-I n-RAEARAEA-c 37 I r.c.KADA16-I n-KADAKADAKADAKADA-c 38 I β KADA8-I n-KADAKADA-c 39 I r.c.EAH16-II n-AEAEAHAHAEAEAHAHA-c 40 II β EAH8-II n-AEAEAHAHA-c 41 II r.c.EFK16-II n-FEFEFKFKFEFEFKFK-c 42 II β EFK8-II n-FEFKFEFK-c 43 I βELK16-II n-LELELKLKLELELKLK-c 44 II β ELK8-II n-LELELKLK-c 45 II r.c.EAK16-II n-AEAEAKAKAEAEAKAK-c 46 II β EAK12 n-AEAEAEAEAKAK-c 47 IV/IIα/β EAK8-II n-AEAEAKAK-c 48 II r.c. KAE16-IV n-KAKAKAKAEAEAEAEA-c 49 IVβ EAK16-IV n-AEAEAEAEAKAKAKAK-c 50 IV β RAD16-IV n-RARARARADADADADA-c 51IV β DAR16-IV n-ADADADADARARARAR-c 52 IV α/β DAR16-IV*n-DADADADARARARARA-c 53 IV α/β DAR32-IV n-(ADADADADARARARAR)-c 52 IV α/βEHK16 n-HEHEHKHKHEHEHKHK-c 54 N/A r.c. EKH8-I n-HEHEHKHK-c 55 N/A r.c.VE20* n-VEVEVEVEVEVEVEVEVEVE-c 56 N/A β RF20* n-RFRFRFRFRFRFRFRFRFRF-c57 N/A β β denotes beta-sheet; α denotes alpha-helix; r.c. denotesrandom coil; N/A denotes not applicable. *Both VE20 and RF20 form abeta-sheet when they are incubated in a solution containing NaCl;however, they do not self-assemble to form macroscopic scaffolds.

The peptides described herein can be chemically synthesized usingstandard chemical synthesis techniques. In some embodiments the peptidesare chemically synthesized by any of a number of fluid or solid phasepeptide synthesis techniques known to those of skill in the art. Solidphase synthesis in which the C-terminal amino acid of the sequence isattached to an insoluble support followed by sequential addition of theremaining amino acids in the sequence is a preferred method for thechemical synthesis of the polypeptides of this invention. Techniques forsolid phase synthesis are well known to those of skill in the art andare described, for example, by Barany and Merrifield (1963) Solid-PhasePeptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.;Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewartet al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill.

II. Antigens

The term “antigen” as used herein refers to a molecule against which asubject can initiate a humoral and/or cellular immune response. Antigenscan be any type of biologic molecule including, for example, simpleintermediary metabolites, sugars, lipids, and hormones as well asmacromolecules such as complex carbohydrates, phospholipids, nucleicacids and proteins. Common categories of antigens include, but are notlimited to, viral antigens, bacterial antigens, fungal antigens,protozoa and other parasitic antigens, tumor antigens, antigens involvedin autoimmune disease, allergy and graft rejection, and othermiscellaneous antigens. In certain compositions and methods of theinvention, the antigen is a peptide.

Viral Antigens. Examples of viral antigens include, but are not limitedto, retroviral antigens such as retroviral antigens from the humanimmunodeficiency virus (HIV) antigens such as gene products of the gag,pol, and env genes, the Nef protein, reverse transcriptase, and otherHIV components; hepatitis viral antigens such as the S, M, and Lproteins of hepatitis B virus, the pre-S antigen of hepatitis B virus,and other hepatitis, e.g., hepatitis A, B. and C, viral components suchas hepatitis C viral RNA; influenza viral antigens such as hemagglutininand neuraminidase and other influenza viral components; measles viralantigens such as the measles virus fusion protein and other measlesvirus components; rubella viral antigens such as proteins E1 and E2 andother rubella virus components; rotaviral antigens such as VP7sc andother rotaviral components; cytomegaloviral antigens such as envelopeglycoprotein B and other cytomegaloviral antigen components; respiratorysyncytial viral antigens such as the RSV fusion protein, the M2 proteinand other respiratory syncytial viral antigen components; herpes simplexviral antigens such as immediate early proteins, glycoprotein D, andother herpes simplex viral antigen components; varicella zoster viralantigens such as gpI, gpII, and other varicella zoster viral antigencomponents; Japanese encephalitis viral antigens such as proteins E,M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80% E, and other Japanese encephalitisviral antigen components; rabies viral antigens such as rabiesglycoprotein, rabies nucleoprotein and other rabies viral antigencomponents. See Fundamental Virology, Second Edition, e's. Fields, B. N.and Knipe, D. M. (Raven Press, New York, 1991) for additional examplesof viral antigens.

Bacterial Antigens. Bacterial antigens which can be used in thecompositions and methods of the invention include, but are not limitedto, pertussis bacterial antigens such as pertussis toxin, filamentoushemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and otherpertussis bacterial antigen components; diptheria bacterial antigenssuch as diptheria toxin or toxoid and other diphtheria bacterial antigencomponents; tetanus bacterial antigens such as tetanus toxin or toxoidand other tetanus bacterial antigen components; streptococcal bacterialantigens such as M proteins and other streptococcal bacterial antigencomponents; gram-negative bacilli bacterial antigens such aslipopolysaccharides and other gram-negative bacterial antigencomponents; Mycobacterium tuberculosis bacterial antigens such asmycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secretedprotein, antigen 85A and other mycobacterial antigen components;Helicobacter pylori bacterial antigen components; pneumococcal bacterialantigens such as pneumolysin, pneumococcal capsular polysaccharides andother pneumococcal bacterial antigen components; hemophilus influenzabacterial antigens such as capsular polysaccharides and other hemophilusinfluenza bacterial antigen components; anthrax bacterial antigens suchas anthrax protective antigen and other anthrax bacterial antigencomponents; rickettsiae bacterial antigens such as romps and otherrickettsiae bacterial antigen component. Also included with thebacterial antigens described herein are any other bacterial,mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.

Fungal Antigens. Fungal antigens which can be used in the compositionsand methods of the invention include, but are not limited to, Candidafungal antigen components; histoplasma fungal antigens such as heatshock protein 60 (HSP60) and other histoplasma fungal antigencomponents; cryptococcal fungal antigens such as capsularpolysaccharides and other cryptococcal fungal antigen components;coccidiodes fungal antigens such as spherule antigens and othercoccidiodes fungal antigen components; and tinea fungal antigens such astrichophytin and other coccidiodes fungal antigen components.

Parasite Antigens. Examples of protozoa and other parasitic antigensinclude, but are not limited to, plasmodium falciparum antigens such asmerozoite surface antigens, sporozoite surface antigens,circumsporozoite antigens, gametocyte/gamete surface antigens,blood-stage antigen pf 1 55/RESA and other plasmodial antigencomponents; toxoplasma antigens such as SAG-1, p30 and other toxoplasmaantigen components; schistosomae antigens such asglutathione-S-transferase, paramyosin, and other schistosomal antigencomponents; leishmania major and other leishmaniae antigens such asgp63, lipophosphoglycan and its associated protein and other leishmanialantigen components; and trypanosoma cruzi antigens such as the 75-77 kDaantigen, the 56 kDa antigen and other trypanosomal antigen components.

Tumor antigens. Tumor antigens which can be used in the compositions andmethods of the invention include, but are not limited to, telomerasecomponents; multidrug resistance proteins such as P-glycoprotein;MAGE-1, alpha fetoprotein, carcinoembryonic antigen, mutant p53,immunoglobulins of B-cell derived malignancies, fusion polypeptidesexpressed from genes that have been juxtaposed by chromosomaltranslocations, human chorionic gonadotrpin, calcitonin, tyrosinase,papillomavirus antigens, gangliosides or other carbohydrate-containingcomponents of melanoma or other tumor cells. It is contemplated by theinvention that antigens from any type of tumor cell can be used in thecompositions and methods described herein.

Antigens Relating to Autoimmunity. Antigens involved in autoimmunediseases, allergy, and graft rejection can be used in the compositionsand methods of the invention. For example, an antigen involved in anyone or more of the following autoimmune diseases or disorders can beused in the present invention: diabetes mellitus, arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemiclupus erythematosis, autoimmune thyroiditis, dermatitis (includingatopic dermatitis and eczematous dermatitis), psoriasis, Sjogren'sSyndrome, including keratoconjunctivitis sicca secondary to Sjogren'sSyndrome, alopecia areata, allergic responses due to arthropod bitereactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drugeruptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves opthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, and interstitial lungfibrosis. Examples of antigens involved in autoimmune disease includeglutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basicprotein, myelin proteolipid protein, acetylcholine receptor components,thyroglobulin, and the thyroid stimulating hormone (TSH) receptor.Examples of antigens involved in allergy include pollen antigens such asJapanese cedar pollen antigens, ragweed pollen antigens, rye grasspollen antigens, animal derived antigens such as dust mite antigens andfeline antigens, histocompatiblity antigens, and penicillin and othertherapeutic drugs. Examples of antigens involved in graft rejectioninclude antigenic components of the graft to be transplanted into thegraft recipient such as heart, lung, liver, pancreas, kidney, and neuralgraft components. An antigen can also be an altered peptide liganduseful in treating an autoimmune disease.

Examples of miscellaneous antigens which can be can be used in thecompositions and methods of the invention include endogenous hormonessuch as luteinizing hormone, follicular stimulating hormone,testosterone, growth hormone, prolactin, and other hormones, drugs ofaddiction such as cocaine and heroin, and idiotypic fragments of antigenreceptors such as Fab-containing portions of an anti-leptin receptorantibody.

III. Pharmaceutical Compositions

The present invention includes methods for preventing or amelioratingmicrobial infections. As such, the invention contemplates vaccines andtherapeutics for use in active immunization of subjects. Immunogeniccompositions can include a peptide fibril coupled to a plurality ofantigens, “fibril complex.”

The preparation of vaccines that contain polypeptide or peptidesequence(s) as active ingredients is generally well understood in theart, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;4,599,230; 4,596,792; and 4,578,770, all of which are incorporatedherein by reference. Typically, such vaccines are prepared asinjectables either as liquid solutions or suspensions: solid formssuitable for solution in or suspension in liquid prior to injection mayalso be prepared. The preparation may also be emulsified. The activeimmunogenic ingredient is often mixed with excipients that arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine may contain amounts of auxiliary substances such as wettingor emulsifying agents, pH buffering agents, or adjuvants that enhancethe effectiveness of the vaccines. In specific embodiments, vaccines areformulated with a combination of substances, as described in U.S. Pat.Nos. 6,793,923 and 6,733,754, which are incorporated herein byreference.

Vaccines and therapeutics may be conventionally administeredparenterally, by injection, for example, either subcutaneously orintramuscularly. Additional formulations which are suitable for othermodes of administration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude, for example, polyalkalene glycols or triglycerides: suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10%, preferably about 1%to about 2%. Oral formulations include such normally employed excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain about 10% to about 95% of active ingredient,preferably about 25% to about 70%.

The compositions described herein may be formulated into apharmaceutical composition as neutral or salt forms.Pharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the peptide) and those that areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

Typically, compositions are administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective and immunogenic. The quantity to be administered depends onthe subject to be treated, including the capacity of the individual'simmune system to synthesize antibodies and the degree of protectiondesired. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner. However,suitable dosage ranges are of the order of several hundred microgramsactive ingredient per vaccination. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by subsequent inoculations or otheradministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection and the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize and health of the subject.

The compositions and related methods of the present invention,particularly administration of a peptide fibril/antigen complex may alsobe used in combination with the administration of traditional therapies.These include, but are not limited to, the administration of antibioticssuch as streptomycin, ciprofloxacin, doxycycline, gentamycin,chloramphenicol, trimethoprim, sulfamethoxazole, ampicillin,tetracycline or various combinations of antibiotics.

In one aspect, it is contemplated that a peptide fibril/antigen vaccineand/or therapy is used in conjunction with antibacterial treatment.Alternatively, the therapy may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agents and/or a proteins is administered separately, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and antigeniccomposition would still be able to exert an advantageously combinedeffect on the subject. In such instances, it is contemplated that onemay administer both modalities within about 12-24 h of each other and,more preferably, within about 6-12 h of each other. In some situations,it may be desirable to extend the time period for administrationsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Various combinations may be employed, for example antibiotic therapy is“A” and the immunogenic composition is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the immunogenic compositions of the present inventionto a patient/subject will follow general protocols for theadministration of such compounds, taking into account the toxicity, ifany. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, suchas hydration, may be applied in combination with the described therapy.

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects of the present invention involveadministering an effective amount of a composition to a subject. In someembodiments of the present invention, immunogenic compositions may beadministered to the patient to protect against infection by one or moremicrobial pathogens. Additionally, such compounds can be administered incombination with an antibiotic or other known anti-microbial therapy.Such compositions will generally be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated. Supplementary active ingredients, such as otheranti-cancer agents, can also be incorporated into the compositions.

In addition to the compounds formulated for parenteral administration,such as those for intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g., tablets or other solidsfor oral administration; time release capsules; and any other formcurrently used, including creams, lotions, mouthwashes, inhalants andthe like.

The active compounds of the present invention can be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular, sub-cutaneous, or even intraperitonealroutes. Typically, such compositions can be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable for useto prepare solutions or suspensions upon the addition of a liquid priorto injection can also be prepared; and, the preparations can also beemulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The proteinaceous compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion, and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Administration of the compositions according to the present inventionwill typically be via any common route. This includes, but is notlimited to oral, nasal, or buccal administration. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, intranasal, or intravenous injection. Incertain embodiments, a vaccine composition may be inhaled (e.g., U.S.Pat. No. 6,651,655, which is specifically incorporated by reference).Such compositions would normally be administered as pharmaceuticallyacceptable compositions that include physiologically acceptablecarriers, buffers or other excipients.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered, if necessary, and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in isotonic NaCl solution andeither added to hypodermoclysis fluid or injected at the proposed siteof infusion, (see for example, Remington's Pharmaceutical Sciences,1990). Some variation in dosage will necessarily occur depending on thecondition of the subject. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject.

An effective amount of therapeutic or prophylactic composition isdetermined based on the intended goal. The term “unit dose” or “dosage”refers to physically discrete units suitable for use in a subject, eachunit containing a predetermined quantity of the composition calculatedto produce the desired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the protection desired.

Precise amounts of the composition also depend on the judgment of thepractitioner and are peculiar to each individual. Factors affecting doseinclude physical and clinical state of the subject, route ofadministration, intended goal of treatment (alleviation of symptomsversus cure), and potency, stability, and toxicity of the particularcomposition.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeutically orprophylactically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above.

IV. Examples

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

EXAMPLE 1 Peptide Fibrils as Immune Adjuvants A. Results

Peptide Design and Supramolecular Assembly. The peptide Q11 waspreviously designed as a self-assembling transglutaminase substrate(Collier and Messersmith, 2003) and was a variation on the DN1 peptideoriginally described by Aggeli and coworkers (Aggeli et al., 1997; Rileyet al., 2009). A peptide containing a Q11 self-assembling domain wasdesigned to include in tandem an OVA₃₂₃₋₃₃₉, a 17-amino acid peptidefrom chicken egg ovalbumin (FIG. 1). OVA₃₂₃₋₃₃₉ (ISQAVHAAHAEINEAGR (SEQID NO:26), hereafter referred to as OVA) is an H-2^(b)-restricted classII peptide containing multiple antigenic determinants, including known Tand B cell epitopes (Yang and Mine, 2009). The self-assembling epitopepeptide (O-Q11) was produced by solid phase synthesis and included ahydrophilic Ser-Gly-Ser-Gly spacer between the OVA and Q11 domains, withthe OVA domain positioned at the N-terminus (FIG. 1B).

When dissolved in water at concentrations of 40 mM and lower, O-Q11formed no visible precipitate. However, similar to Q11 and to otherpreviously reported Q11 derivatives (Collier, 2008; Collier andMessersmith, 2003; Jung et al., 2008), it formed networks of laterallyentangled fibrils when it was first dissolved in water and then added tosalt-containing buffers such as phosphate-buffered saline (PBS), (FIG.1A, FIG. 2). By TEM, O-Q11 in PBS appeared as long, unbranched fibrilswith widths of about 15 nm (FIG. 2C). Although the length of thesefibrils was not directly measured, the relative scarcity of fibril endsin TEM images suggested that the fibril length was on the order ofmicrons. Circular dichroism of O-Q11 showed a singleconcentration-dependent minimum at 229-232 nm (FIG. 2E). This spectrumis consistent with a high degree of β-sheet or β-turn structure, and itis similar to the spectra of Q11 and mixtures of Q11 with otherpreviously reported ligand-bearing Q11 derivatives (Jung et al., 2009;Collier and Messersmith, 2003).

Epitopes were functionally displayed on O-Q11 fibrils. The availabilityof the OVA epitope on the surface of O-Q11 nanofibers was confirmed byTEM and by ELISA. To label epitopes in TEM samples, an N-terminallybiotinylated O-Q11 was synthesized with a single biotin tag directlyadjacent to the OVA sequence. This biotinylated peptide produced fibrilsthat appeared morphologically similar to O-Q11 (FIG. 2D). Five-nanometerstreptavidin-conjugated gold particles were then used to probe epitopeavailability on the fibril surface, with unmodified Q11 serving as anegative control. Biotin-O-Q11 fibrils stained strongly withstreptavidin-gold, whereas Q11 samples bound negligible numbers ofparticles (FIG. 2B-2D), demonstrating that a significant portion of thepeptides' N-termini were available on the surface of the fibrils. Toconfirm this finding and to quantify the availability of the entireepitope, ELISA was employed. Plates coated with OVA and O-Q11 peptideswere probed with antisera from mice immunized with OVA, either with orwithout complete Freund's adjuvant (CFA). Antisera from CFA-adjuvantedgroups showed similar titers of OVA-reactive IgG, whether measured onOVA plates or on O-Q11 plates, and both showed low backgrounds forantisera from non-adjuvanted groups (FIG. 2F). This result indicatedthat the OVA epitope was functionally presented on the surface of theQ11 fibrils, and that plates coated with O-Q11 fibrils produced similarsignal strengths to plates coated with the non-fibrillized OVA peptide,allowing the sera of mice immunized with different peptides to becompared. Slightly higher titers were observed for the O-Q11-coatedplates in both cases, but it was not statistically significant.

High IgG titers were elicited by O-Q11 without adjuvant in mice. Toinvestigate how fibrillization affected the immunogenicity of OVA,C57BL/6 mice were immunized subcutaneously with the different peptidesand boosted with additional peptide at 28 days (see methods). Serum wascollected seven days after the boost, and levels of variousimmunoglobulins were measured. It was found that fibrillized Q11 alonedid not raise any detectable IgG, whether delivered with or without CFA(FIG. 3A-3B). This result reiterated previous findings that Q11 was notimmunogenic (Jung et al, 2009) and further indicated that Q11 continuedto be non-immunogenic even when delivered in CFA. Also, Q11 andfunctionalized Q11 peptides did not induce cell death in cultures ofprimary human endothelial cells, indicating that the basic Q11 sequencewas non-cytotoxic as well as non-immunogenic (Jung et al., 2009; Jung etal., 2008).

In surprising contrast to the immeasurably low immunogenicity of Q11,O-Q11 elicited high IgG titers without any added adjuvant (FIG. 3A).Even higher titers were produced when it was delivered in CFA (FIG. 3B).Anti-peptide IgG titers were similar between mice injected with O-Q11 inPBS and OVA peptide in CFA, indicating that the Q11 sequence itselffunctioned as a strong adjuvant. O-Q11 antisera also bound to OVA-coatedplates, demonstrating that the epitope was conserved (FIG. 3C). O-Q11antiserum reactivity to OVA-coated plates also excluded the possibilitythat the high antibody titers found in O-Q11 antisera were a measurementartifact arising from increased antigen density on fibril-coated ELISAplates. In addition, endotoxin levels were less than 0.3 EU/mL for allsamples (Table 2), eliminating the possibility that inadvertentcontamination caused the observed adjuvant effect. O-Q11 antisera alsoreacted with Q11, though at smaller, statistically insignificant levels(FIG. 3C), possibly indicating a small degree of epitope spreading tothe Q11 domain.

TABLE 2 Purity (%) Endotoxin (m/z) (m/z) by HPLC^(a) (EU/mL)^(a,b)calc'd found^(a) Q11 92.0-97.5% 0.024-0.098 1526.7 1527.2-1529.4OVA₃₂₃₋₃₃₉  91.6-95%  bkgd-0.112 1773.9 1774.0-1774.3 O-Q11 96.2-98.0%0.08-0.28 3528.8 3528.3-3530.1 ^(a)Ranges represent high and low valuesamong three batches for each peptide. ^(b)All are within acceptablelimits [Malyala P, Singh M (2008), Endotoxin limits in formulations forpreclinical research J Pharm Sci 97, 2041-2044].

It is contemplated that the multivalent surface display of the epitopeon the fibrils was the source of Q11's strong adjuvant properties.Alternatively, if Q11 functioned as an adjuvant by activating Toll-likereceptors, similarly to LPS or unmethylated CpG motifs, or if it simplyslowed the diffusion of the epitope from the injection site bysurrounding it with fibrils (the so-called “depot effect”), thenoutright conjugation of the epitope and the fibril would not berequired. Additionally, if Q11 fibrils functioned in a manner similar toparticulate adjuvants such as aluminum salts, whereby the adsorption andentrapment of the antigen onto and within the particle is sufficient foradjuvancy, then conjugation would also not be required. To investigatethis aspect, mice were injected with unconjugated mixtures of Q11peptide and OVA peptide. Notably, the strong antibody response generatedby O-Q11 was completely abolished in the absence of covalent couplingbetween the Q11 fibrillizing domain and the epitope domain (FIG. 3D). Nodetectable IgG was observed for mixtures of OVA and Q11. This resultindicated that Q11's adjuvant properties were dependent on its covalentattachment to the epitope peptide.

To determine the nature of the immune response to Q11-adjuvantedpeptides, the isotypes of the responding antibodies were evaluated. Forboth OVA in CFA and fibrillized O-Q11, the dominant antibody isotype wasIgG1, with smaller amounts of IgG2a, IgG2b, IgG3, and IgM being producedfor both (FIG. 4). Comparing CFA-adjuvanted responses withQ11-adjuvanted responses, IgG1, IgG2a, and IgG3 were produced instatistically similar quantities, but IgG2b production was greater inthe CFA-adjuvanted group, and IgM production was greater in theQ11-adjuvanted group.

To investigate the involvement of T cell help in the immune responses toO-Q11, splenocytes from immunized mice were challenged in vitro with thepeptides, and the production of interferon-γ (IFN-γ), interleukin-2(IL-2), and interleukin-4 (IL-4) was measured. These three cytokineswere selected to provide measures of either a Th1 response (IFN-γ andIL-2) or a Th2 response (IL-4). However, somewhat surprisingly, whensplenocytes from immunized mice were challenged in vitro with theimmunizing peptide, they did not produce significant levels of any ofthese three cytokines compared with positive controls (FIG. 5).

Attaching a short self-assembling amino acid sequence to a peptideepitope's C-terminus can dramatically enhance the peptide'simmunogenicity. The results indicate that fibrillizing peptide domainsmay be useful as simple adjuvant systems for peptide-basedimmunotherapies, and these results also provide guidance for thedevelopment of non-immunogenic peptide biomaterials within applicationssuch as regenerative medicine. As a means to specifically enhancepeptide immunogenicity, the novel Q11-based approach reported here has anumber of non-limiting advantages. For example, a self-assemblingpeptide domain can be easily added to any known epitope usingconventional solid phase peptide synthesis. Also, by utilizingself-assembly, highly multivalent nanoscale objects can be directlyproduced from only one molecule, which enables precision in theproduction, purity, and study of the material. In contrast, manyadjuvants currently employed or under development are comprised ofmultiple molecular constituents or heterogeneous mixtures, making theirdefinition, formulation, purification, and characterization challenging.For example, immunotherapies based on attenuated live viruses orheat-killed organisms contain intrinsic adjuvants such aslipopolysaccharide or unmethylated CpG motifs that are critical to theirefficacy (McKee et al., 2007). Adjuvants based on natural products suchas saponins or squalene (Sun et al., 2009) are by nature associated withsome degree of molecular heterogeneity, and particulate adjuvants suchas aluminum salts depend on antigen adsorption or entrapment, processeswhich are complexly dependent on multiple chemical and physical factorsduring formulation (Marrack et al., 2009; McKee et al., 2007).Accordingly, specific molecular features of current adjuvants are noteasily adjusted independently within a given vaccine formulation, makingit difficult both to optimize an immunotherapy as well as understand itsmechanism of action. Such efforts will be greatly enhanced with theavailability of chemically defined adjuvants.

B. Materials and Methods

Peptide Synthesis and Purification. Peptides were synthesized usingstandard Fmoc chemistry as previously reported (Jung et al., 2009; Junget al., 2008). For TEM studies, O-Q11 was N-terminally biotinylatedon-resin using biotin o-nitrophenyl ester. Peptides were purified usinga Varian ProStar HPLC system, Grace-Vydac C18 reverse phase columns, andwater-acetonitrile gradients. Peptide identity and purity were confirmedby MALDI-MS and HPLC, respectively (Table 2). Endotoxin levels of allimmunizations were <0.3 EU/mL by LAL chromogenic endpoint assay (Lonza),well within acceptable limits (Table 2).

Circular Dichroism. An AVIV 202 CD spectrometer (Aviv Biomedical, N.J.),0.1 cm path length quartz cells, and initial disaggregation in TFA wereemployed as previously reported (Jung et al., 2009). Workingconcentrations were prepared in degassed water using Phe absorbance at257 nm. Owing to peptide fibrillization and the resultant low CD signalstrength, spectra below 220 nm could not be measured accurately and soare not reported.

Transmission Electron Microscopy. Peptides were dissolved in deionizedwater and mixed 6:1 with PBS to produce working peptide concentrationsof 330 μM. After fibrillizing for 4 h, peptides were applied to 400 meshgold grids with carbon support films, negative-stained with 1% uranylacetate, and imaged on a Tecnai F30 TEM. For gold staining, prior tonegative-staining grids were placed upside-down on a droplet of blockingsolution (0.2% acetylated BSA, 0.1% gelatin from cold water fish skin inPBS) for 5 min, then for 2 h on a droplet of 5 nm colloidal goldconjugated to streptavidin (Sigma). Grids were washed once with blockingsolution, twice with PBS, and stained with 1% uranyl acetate.

Immunizations. Peptides were dissolved in sterile water (8 mM) andallowed to fibrillize overnight at 4° C. Stock solutions were thendiluted in sterile, endotoxin-free PBS to working concentrations. FemaleC57BL/6 mice (6-8 weeks old, Taconic Farms, Ind.) were each given two 50μL subcutaneous injections near the shoulder blades, each injectioncontaining 100 nmol of peptide. CFA-adjuvanted groups received the samevolume and total peptide dose, prepared by emulsifying peptide/PBSsolutions 1:1 in CFA. Mice were boosted at 28 days with two additional25 μL injections, each containing 50 nmol of peptide. CFA groups wereboosted in incomplete Freund's adjuvant (IFA). Mice receiving mixturesof Q11 and OVA received 100 nmol of each peptide in the first injectionand 50 nmol of each in the second. Seven days after the boost, the micewere sacrificed, and sera and spleens were harvested. In all animalwork, institutional guidelines for the care and use of laboratoryanimals were strictly followed under a protocol approved by theUniversity of Chicago's Institutional Animal Care and Use Committee.

Determination of antibody titers. High-binding ELISA plates(eBioscience) were coated with either 20 μg/mL peptide in PBS or PBSalone (for uncoated background subtraction) overnight at 4° C. Wellswere blocked with 1% BSA/0.5% Tween 20 in PBS, and serial dilutions ofserum between 1:10² and 1:10⁹ were applied, followed byperoxidase-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch). Washing steps were performed with 0.5% Tween 20 in PBS,plates were developed using TMB substrate (eBioscience), and absorbancevalues were read at 450 nm. To determine titers for each antiserum,background absorbance values from uncoated wells were subtracted fromcoated wells, and net absorbances were compared to cutoff values. Thecutoff consisted of the mean plus three times the standard deviation ofthe negative control group (mice receiving OVA without adjuvant) foreach corresponding dilution. Any sample dilutions having absorbancesabove this cutoff value were considered positive readings. The titer wasconsidered as the highest dilution for which it and all lower dilutionshad positive readings. If no positive dilutions were present the titerwas considered to be 10². Negative control mice (OVA without adjuvant)did not raise detectable IgG, and no single mouse in the negativecontrol groups had absorbance values greater than three standarddeviations above the group's mean for a given dilution; therefore allnegative control mice are reported as having titers of 10², which is thebaseline level of detection for this study. Antibody isotypes wereanalyzed similarly using a mouse monoclonal antibody kit containing goatanti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgM (Sigma).

Splenocyte isolation and challenge. Spleens of the immunized mice werepressed through 70 μm cell strainers, and isolated splenocytes werewashed in RPMI medium containing 10% FBS. Red blood cells were lysedusing ACK buffer (150 mM NH₄Cl, 10 mM KHCO₃/0.1 mM EDTA) and washedtwice. 1×10⁶ cells/well (96 well plate) were plated in complete T cellmedium (S-MEM supplemented with 3.75 mM dextrose, 0.9% L-glutamine, 0.6%essential amino acids, 1.26% non-essential amino acids, 0.9% sodiumpyruvate, 9 mM sodium bicarbonate, 95 μM gentamycin, 140 μMpenicillin-G, 60 μM streptomycin sulfate, 44 μM 2-mercaptoethanol, and10% fetal bovine serum) containing 5 μg/ml challenging peptide or nopeptide. After a 24 h, interferon-γ (IFN-γ), IL-2, and IL-4concentrations were measured in the culture medium using a sandwichELISA. Antibodies and reagents were purchased from eBioscience, andcytokine concentrations were calculated from standard curves.

Statistical Analysis. Statistical analysis was performed by ANOVA withTukey's HSD post hoc comparisons. The data reported in FIGS. 3A and FIG.4 represent three separate experiments, each containing five mice pergroup. Positive and negative controls were not statistically differentbetween the three trials, and so the data were pooled into the groups of15 shown.

EXAMPLE 2 Characterization of Immune Responses to Q11 Linked Peptides A.Results

Antibody responses to self-assembled peptide antigens are long-lived. Ina previous study it was shown that conjugating an antigenic peptide(OVA) to the self-assembling peptide domain Q11 results in robustantibody responses against OVA. It was found that mice immunized with100 nmol of the fibrillized antigen elicited similar antibody titers asthe free peptide administered in complete Freund's adjuvant (CFA). Thedose dependence and longevity of the antibody response to OVA-Q11 wasfurther investigated and antibody responses against the malarial peptideepitope (NANP)₃ coupled to Q11 were also characterized. Mice wereimmunized with two different doses of OVA-Q11 (100 nmol and 50 nmol), or100 nmol of (NANP)₃-Q11 and boosted with half the dose at wk 4. Micewere bled on a weekly basis up to wk 6, once at wk 8, and once everyfour weeks thereafter.

Serum ELISAs indicated that antibody production was long-lived, asantibodies were detectable for at least 36 wks in OVA-Q11-immunizedmice. Similar levels were detected in OVA peptide delivered in CFA. Theantibody levels spiked approximately 2.5 fold after the boost at wk 4and reached maximum levels around wk 6 in the OVA-Q11 immunized mice forboth the 100 nmol and 50 nmol groups. Although the antibody productionat earlier time points (wk 3 and wk 4) was higher in the 100 nmol group,no significant difference was observed after the boost and throughoutthe duration of the study. This suggested that immunizing mice with 50nmol of OVA-Q11 elicited antibody responses with similar magnitude andlongevity as mice immunized with 100 nmol of OVA-Q11.

Mice immunized with (NANP)₃-Q11 also elicited sustained antibodyresponses that were detectable for up to 28 wks; however, unlike OVA-Q11no spike in antibody levels was observed after the boost. These resultsdemonstrate that self-assembled peptides decorated with peptide antigenscan elicit long term antibody production in mice without the need forfrequent boosting.

Co-assembled peptide antigens elicit immune responses independently.Transmission electron microscopy showed that OVA-Q11, (NANP)₃-Q11, andco-assembled OVA-Q11/(NANP)₃-Q11 self-assembled into fibers in phosphatebuffered saline. To investigate if antibodies were raised simultaneouslyagainst multiple self-assembled antigens co-administered in vivo, micewere immunized with OVA-Q11 and (NANP)₃-Q11 either separately or asco-assembled fibrils. The total peptide concentration was the same inboth groups. Injecting the peptides either separately or as co-assembledfibrils, resulted in production of antibodies against both epitopes.Antibodies were produced for up to 16 wks with similar magnitudes,suggesting that the presence of one peptide did not alter immuneresponses towards the other. Interestingly, the spike in the antibodylevels of OVA-Q11 at wk 5 was not observed. However, there was a smallincrease in the antibody levels in NANP-Q11 boosted mice at wk 5 in bothgroups, which was not detected when mice were immunized with (NANP)₃-Q11alone.

Antibodies generated against self-assembled peptides cross-reacted withwhole proteins. For efficient protection against disease antibodiesraised against single epitopes must react with and neutralize wholeproteins or pathogens. To determine if the antibodies produced againstself-assembled OVA peptide reacted with whole ovalbumin, serum derivedfrom OVA-Q11 immunized mice was applied to ELISA plates coated withOVA-Q11 or ovalbumin. Antibodies raised against OVA-Q11 were reactiveagainst ovalbumin.

Peptide Assemblies were observed in the lymph nodes for extended timeperiods. Immunohistochemistry data and staining for biotin on thepeptides indicated that OVA-Q11 was localized to the draining lymphnodes over time. Peptide assemblies (stained brown and indicated byarrows) were found at the cortex of the lymph nodes by wk 1 andcontinued to persist up to wk 6. No peptide was observed at time pointsearlier than one week (1 day and 3 days). Although the mechanism oftrafficking is unknown, co-staining for macrophages, dendritic cells, orB cells might provide further insights into the uptake and processing ofthese peptides in vivo.

Antibody responses to self-assembled peptides are T cell-dependent. Therobust antibody responses and isotype class switching observed withOVA-Q11 suggested involvement of CD4+T cells. To determine therequirement for T cells, an adoptive T cell transfer assay wasperformed. Specifically, mice were injected with CFSE labeled OTIIcells, which express receptors for the H-2^(b) restricted OVA epitope.Antigen-mediated proliferation causes a decrease in the CFSE intensitywhich can be detected by flow cytometry.

Robust proliferation of OTII cells in the OVA-Q11 and OVA-CFA immunizedmice was observed by the shift in the CFSE intensity peak whichdemonstrated that T cells were involved in the immune responses againstOVA-Q11. Quantifying the shift in the CFSE intensity indicated that morethan 95% of the transferred cells in both OVA-Q11 and OVA-CFA immunizedmice proliferated. In contrast, a negligible shift in CFSE intensity wasobserved for Q11-immunized mice. The extent of proliferation was foundto be similar in both the spleen and the lymph nodes. Immunization withOVA-Q11 after adoptive transfer of OTII cells elicited rapid antibodyproduction compared to mice that were not given OTII cells.Interestingly, mice that were injected with OTII cells and immunizedOVA-CFA failed to generate antibody responses within the same timeframe.

To confirm the necessity of T cells, KO mice lacking both the αβ and γδreceptors for CD4⁺ T cells were immunized with OVA-Q11 along with wildtype controls. No antibody production was observed in mice lacking the Tcell receptors over 6 wks suggesting that T cells are required forantibody production against OVA-Q11. The wild type control miceresponded as expected. Together, these data support the earlier T cellrequirement conclusion. To ensure that the KO mice are capable ofeliciting immune responses, they were immunized with a T-independentantigen, NP-Ficoll. Antibody production against NP-Ficoll was observedin the KO mice demonstrating their capability to mount B cell-mediatedimmune responses and reconfirming that antibody responses againstOVA-Q11 were T cell-mediated. This finding might not be surprisingbecause OVA is a strong MHC class II peptide, containing both B and Tcell determinants. To further determine if T cells are required forother epitopes, T cell receptor KO mice were immunized with (NANP)₃-Q11which resulted in no antibody production. These findings suggest thatCD4⁺ T cells might play an important role in the immune responsesagainst self-assembled antigens.

B. Materials and Methods

Peptide Synthesis and Purification Peptides were synthesized on aCSBio136-XT peptide synthesizer using standard Fmoc chemistry, cleavedusing cocktail solution (95% trifluoroacetic acid, 2.5% water and 2.5%triisopropyl silane), precipitated in diethyl ether, and lyophilized.Peptides were purified using a Varian ProStar HPLC system, Grace-VydacC18 reverse phase columns, and water/acetonitrile gradients. Theacetonitrile was removed by centrifugal evaporation and the peptideswere lyophilized and stored at −20° C. until further use. Peptideidentity and purity were confirmed by MALDI-MS and HPLC, respectively.Endotoxin levels of all formulations were tested using a LimulusAmebocyte Lystae (LAL) chromogenic end point assay (Lonza, USA).Endotoxin levels in all immunization formulations were found to be lessthan <0.22 EU/mL. The peptides synthesized wereNANPNANPNANPSGSGQQKFQFQFEQQ ((NANP)₃-Q11 (SEQ ID NO:27)), andISQAVHAAHAEINEAGRSGSGQQKFQFQFEQQ (OVA-Q11 (SEQ ID NO:28)).

Transmission Electron Microscopy Stock solutions of 1 mM peptides wereallowed to fibrillize in PBS for 4 h at room temperature or overnight at4° C., diluted to 0.25 mM, and pipetted onto carbon-coated 200 meshlacey grids (Electron Microscopy Sciences). For the co-assembledfibrils, OVA-Q11 and (NANP)₃-Q11 peptides were mixed as dry powders,dissolved in deionized water, and diluted in PBS to produce workingpeptide concentration of 0.5 mM. After fibrillizing for 4 h, peptideswere applied to the grids. The grids were stained with 1% uranyl acetatefor 2 minutes, and imaged with a FEI Tecnai F30 transmission electronmicroscope.

Animals and Immunizations Peptides were dissolved in sterile water (8 mMstock), incubated overnight at 4° C., and diluted in sterile PBS (2 mMworking concentration) prior to immunizations. For the malaria peptide,(NANP)₃-Q11, the formulations were incubated in PBS overnight to ensurecomplete fibril formation. To prepare co-assembled fibers ofOVA-Q11/(NANP)₃-Q11 or OVA-Q11/Biotin-Q11, the peptides were combined asdry powders, mixed thoroughly, and dissolved in sterile water. Thepeptides were allowed to co-assemble overnight at 4° C. and fibrillizedin sterile PBS. Female C57BL/6 mice (6-8 weeks old, Taconic Farms, Ind.)were immunized subcutaneously with two 50 μL injections in the back,with each injection containing 100 nmol of peptide. Mice were boosted at28 days with two additional 25 μL injections, each containing 50 nmol ofpeptide. Blood was drawn on a weekly basis via the submandibularmaxillary vein, the serum extracted, and stored at −80° C. until use. Toidentify local tissue responses and peptide distribution, mice wereinjected intramuscularly with 50 μL of peptide solution (90% OVA-Q11+10%Biotin-Q11) in each thigh and the muscles and inguinal lymph nodes wereextracted at predetermined time points. In all animal work,institutional guidelines for the care and use of laboratory animals werestrictly followed under a protocol approved by the University ofChicago's Institutional Animal Care and Use Committee.

Determination of Antibody Production ELISA plates (eBioscience) werecoated with 20 μg/mL of peptide or 0.5 μg/mL of whole protein in PBSovernight at 4° C. The plates were blocked with 200 μL of 1% BSA in PBST(0.5% Tween-20 in PBS) for 1 h and serial dilutions of serum between1:10² and 1:10⁹ were applied (100 μL/well) for 1 h at room temperature.Peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch) (1:5000 in 1 BSA-PBST, 100 μL/well) was then applied for 30min and the plates were developed using TMB substrate (100 μL/well,eBioscience). The reaction was stopped using 50 μL of 1 M phosphoricacid and 100 μL of the solution was transferred to fresh plates andabsorbance values were read at 450 nm. Absorbance values of PBS (noantigen) coated wells were subtracted to account for background. Theplates were washed between each step with PBST.

Immunohistochemistry Freshly dissected skeletal muscle and inguinallymph nodes were fixed overnight with 4% formaldehyde inphosphate-buffered saline (pH 7.2) and the tissue was processed andembedded in paraffin at the Human Tissue Resource Center (University ofChicago). The paraffin blocks were cut on a microtome (Leica, USA) into4 μm thick sections and were deparaffinized with xylene and rehydratedwith gradient ethanol. The sections were boiled in 10 mM sodium citratesolution (pH 6.0) for 10 min to retrieve the antigens and blocked with5% BSA in PBS for 1 hr. The sections were then incubated withHorseradish Peroxidase Avidin D (Vector Laboratories, Calif.) for 3 hrs,and the biotin was visualized using a peroxidase substratediaminobenzidine kit (Vector Laboratories, Calif.). The sections werealso counterstained with Mayer's hematoxylin solution (Sigma-Aldrich,USA) for 10 mins. The slides were dehydrated with gradient ethanol andxylene and mounted with Permount Mounting Medium (Fisher Scientific,Pa.). Images were acquired using a Zeiss Axioskop at the Microscopy CoreFacility (University of Chicago).

Adoptive Transfer of T cells OTII transgenic mice on C57BL/6 background,whose T cells recognize the OVA peptide and carry a marker todistinguish them from naïve T cells from B6 mice (CD90.1 congenic). Invivo CD4+ T cell proliferation was analyzed after adoptive transfer intomice and immunization with the peptides. Briefly, splenocytes and lymphnodes of OTII+ C57BL/6 mice were processed into a single cell suspensionand enriched for CD4+ T cells, by negative magnetic separation (MiltenyiBiotec). The cells were then labeled with 10 μM carboxyfluoresceinsuccinimidyl ester (CFSE) and adoptively transferred into mice (5×10⁵OTII cells/mouse, retroorbital injection) 24 h before peptideimmunizations. The next day, mice were immunized with 100 nmol ofOVA-Q11 in PBS, OVA in CFA, or Q11 in PBS at two different sitessubcutaneously. Five days after immunizations, spleens and lymph nodeswere harvested, processed into single cell suspensions, and stained forflow cytometry in 1%BSA in PBS containing 0.02% sodium azide (FACSbuffer) for 1 hour at 4° C. with APC-labeled anti-CD4 (RM 4-5) (BDBiosciences, N.J.) and PECy7-labeled anti-CD90.1 (HIS51) (eBioscience,CA) respectively. Anti-CD16/32 (2.4G2.1) (University of ChicagoImmunology Core) was used to prevent nonspecific antibody binding. Afterstaining, cells were washed twice with FACS buffer and samples were runon the LSR-II (BD Biosciences, N.J.) flow cytometer and analyzed forevidence of proliferation as indicated by dilution of CFSE intensityusing. FlowJo cytometry analysis software (Tree Star, Oreg.) was used toanalyze and quantify the results. Blood was collected through cardiacexsanguination and serum was extracted and stored at −80° C. untilfurther use.

EXAMPLE 3 Modification of Fibril Monomers A. Results.

Synthesis of Phosphonate-Q11. MALDI-TOF-MS analysis demonstrated thatphosphonate-Q11 can be synthesized in good yield by reactingphosphonate-maleimide with cys-terminated Q11. Similar to Q11,phosphonate-Q11 self-assembled into fibrils when dissolved in 1× PBS ata concentration of 0.25 mM.

Cutinase-GFP reacts efficiently with mixed Q11:phosphonate-Q11 fibrils.The reaction of cutinase with phosphonate results in the release ofp-nitrophenol, which absorbs light at 403 nm. The absorbance ofsolutions containing 1 mM Q11:phosphonate-Q11 (99:1 molar ratio) and4.5, 9, or 15 μM cutinase-GFP at 403 nm increased as a function of time.Absorbance values reached a plateau for the 4.5 μM cutinase-GFPcondition by 300 minutes, which suggested that the reaction approachedcompletion over this time frame. For the 9 and 15 μM cutinase-GFPconditions, a time course of nearly 600 minutes was required for thesereactions to approach completion.

To characterize the extent of reaction between cutinase-GFP andphosphonate-Q11, unreacted cutinase-GFP was removed from the peptidefibrils by serial centrifugation washes and then measured thefluorescence intensity of the purified fibrils. When 0.9 μM or 1.8 μMcutinase-GFP was reacted with 1 mM Q11:phosphonate-Q11 (99:1 molarratio), the fluorescence intensity of fibrils was similar to thefluorescence of cutinase-GFP in 1× PBS at the same concentration,suggesting that the reaction proceeds nearly quantitatively under theseconditions. When the cutinase-GFP concentration was increased to 4.5, 9or 15 μM, however, the resulting fibril fluorescence intensity was equalto GPF concentrations of 2.5, 4.12 or 4.75 μM, respectively, whichcorresponds to an extent of reaction of approximately 50% for eachcondition. This suggests that as stoichiometric ratios of cutinase andphosphonate are reached, the extent of reaction is limited byphosphonate moiety availability, which may be influenced by packing ofprotein onto the fibrils (i.e. a steric limit) or burying of phosphonatemoieties within the fibrils during assembly.

The influence of fibril preparation on the reaction betweenphosphonate-Q11 and cutinase-GFP was also characterized. The reactionkinetics and extent of reaction between cutinase-GFP and phosphonate-Q11were similar for two different cutinase-GFP concentrations, regardlessof whether the fibrils were prepared by mixing an aqueous solution ofQ11 with an aqueous solution of phosphonate-Q11 at a 99:1 volume ratioor by dissolving a mixture consisting of a 99:1 molar ratio of dry Q11and phosphonate-Q11, illustrating robustness in formulating thematerials.

Mice immunized with GFP-Q11 fibrils produced antibodies againstcutinase-GFP. CB57BL/6 mice immunized with 3.58 μM GFP-cutinaseconjugated to 1 mM Q11:phosphonate-Q11 fibrils (99:1 molar ratio)produced IgG antibodies that were reactive against microtiter platescoated with GFP-cutinase-phosphonate-Q11 fibrils by 1 weekpost-immunization. The amount of total IgG produced by these miceincreased by week 2, and then decreased at week 3 and 4. Compared tomice injected with GFP-cutinase emulsified in complete Freund'sadjuvant, a commonly used potent adjuvant in mouse models, the totalamount of IgG antibodies produced by mice immunized with GFP-cutinaseconjugated to mixed Q11:phosphonate-Q11 fibrils were lower, however, thetrajectory of antibody production was similar in both groups.Importantly, mice immunized with GFP-cutinase in 1× PBS demonstrated lowlevels of antibody production through 4 weeks, indicating that theobserved antibody production in mice immunized with GFP-cutinaseconjugated to mixed Q11:phosphonate-Q11 fibrils is not due to theinjection of foreign proteins into the mice, but rather requires theaction of an adjuvant.

Mice were boosted with a half dose of GFP-cutinase conjugated toQ11:phosphonate-Q11 fibrils (99:1 molar ratio) at day 31, which resultedin increased IgG antibody levels that were maintained through week 6.The total IgG produced post-boost was similar in mice immunized andboosted with GFP-cutinase conjugated to mixed Q11:phosphonate-Q11fibrils or mice immunized with GFP emulsified in CFA and boosted withGFP-cutinase emulsified in incomplete Freund's adjuvant. This suggestedthat chemically-defined self-assembled fibrils decorated with proteinantigens are as effective as ill-defined oil emulsions as adjuvants forprime-boost vaccination strategies.

To determine the reactivity of antibodies produced by mice immunizedwith GFP-cutinase conjugated to mixed Q11:phosphonate-Q11 fibrils serumcollected at week 5 was reacted with ELISA plates coated withGFP-cutinase-phosphonate-Q11 fibrils or GFP-cutinase. Antibodyreactivity against toward both plates was similar, which indicates thatthe antibodies produced around week 5 are primarily reactive towards theGFP-cutinase fusion protein and are relatively unreactive towards themixed Q11:phosphonate-Q11 adjuvant.

Antibody isotype is an important indicator of the immunological pathwaysinvolved in antibody production. IgG1 was the prominent isotype in serumcollected at week 5 from mice immunized with GFP-cutinase conjugated tomixed Q11:phosphonate-Q11 fibrils, while lower levels of IgG2a, IgG2b,IgG3, or IgM were also present. Interestingly, while IgG1 was also themajor antibody isotype present in serum collected from mice immunizedwith GFP-cutinase emulsified in CFA at week 5, IgG2b was also elevatedcompared to other isotypes. The predominance of IgG1 suggests that forboth adjuvants, antibody production involves Th-2 mediated,IL-4-dependent, B cell isotype switching.

B. Materials and Methods

Cutinase-GFP expression. Origami B (DE3) E. coli transformed with thecutinase-GFP fusion vector were cultured in 10 mL 2XTY media with 100μg/mL carbenicillin and 50 μg/mL kanamycin A overnight at 37° C. on arotary shaker at 2200 rpm. After overnight culture, E. coli weresubcultured into a 1 L flask containing 2XTY media with 100 μg/mLcarbinocillin and 50 kanamycin A. The flask was incubated at 37° C. in arotary shaker at 2200 rpm and the cells were allowed to proliferateuntil reaching an optical density at 600 nm of 0.6-0.8. When the E. colireached an appropriate density, expression of cutinase-GFP was inducedby adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to themedium. IPTG-induced cells were cultured for 16 hours at 18° C. on arotary shaker at 2200 rpm. At the end of the expression period, cellswere pelleted by centrifugation at 9800 rpm for 10 minutes. Media wasdecanted from the pellet and E. coli was washed once by resuspending in400 mL of buffer containing 50 mM Tris-HCl, 3 mM MgCl₂, 250 mM NaCl(Buffer A) followed by centrifugation at 9800 rpm for 10 minutes.Following centrifugation, buffer A was decanted from the pellet, E. coliwas resuspended in 35 mL buffer A, and E. coli was transferred to a 50mL conical tube. E. coli were lysed to release cutinase-GFP by adding3.5 mL 10× BugBuster, 300 units DNAse, and one eComplete mini EDTA-freeprotease tablet to the buffer and incubating for 20 minutes at roomtemperature on a table-top shaker. Cell debris was separated fromGFP-cutinase by centrifuging the lysis buffer at 20000 rpm, 4° C. for 35minutes. His₆-tagged cutinase-GFP was further purified by columnchromatography by passing the centrifugation supernatant over a cobaltcolumn. The column eluent was collected and passed over the column asecond time to increase yield of GFP-cutinase recovered. Protein boundto the cobalt column was washed with 10 column volumes of buffer A.Protein was eluted from the column by passing 50 column volumes ofBuffer A containing 0.5% Triton X-100 over the protein-bound column. Theeluted protein was dialyzed overnight at 4° C. against buffer containing25 mM Tris-HCl, 150 mM NaC1, 0.05% NaN₃, pH 7.8 to exchange salts andremove endotoxin bound to Triton X-100. Protein was concentrated bycentrifugation using an Amicon Ultra centrifugal filter. Proteinmolecular weight was analyzed by SDS-PAGE. GFP activity wascharacterized by exposing the protein to a 395 nm UV light and cutinaseactivity was characterized by incubating protein in 1×phosphate-buffered saline (pH 7.4) containing dinitrophenol andmeasuring absorbance at 405 nm. Protein was stored in buffer at 4° C. or−20° C. until use.

Endotoxin quantification and removal. Endotoxin level in GFP-cutinasewas quantified using a Limulus amebocyte lysate assay according tomanufacturer's instructions. Endotoxin was removed from GFP-cutinase by:(1) adding cold Triton X-114 at a 1:10 (v/v) ratio to a solution ofGFP-cutinase, (2) incubating this solution on ice for 10 minutes, (3)incubating this solution at 37° C. for 20 minutes to precipitateendotoxin-loaded Triton X-114 micelles, (4) centrifuging at 4000 rpm for5 minutes, and (5) collecting the endotoxin-free supernatant containingGFP-cutinase. This process was repeated once to ensure removal ofendotoxin.

Phosphonate-Q11 synthesis—The peptides Q11 or C-SGSG-Q11 weresynthesized on a CSBio 136-XT using a standard solid phase peptidesynthesis protocol based on Fmoc-protected α-amine and HOBt, HBTU, DI KAactivation. Peptide was cleaved from the resin by incubating in acocktail containing 95% trifluoroacetic acid, 2.5% triisopropylsilane,and 2.5% water for 90 minutes. At the end of the cleavage reaction, thecocktail was passed through a filter to separate the peptide from thesynthesis resin. The cocktail was then removed using a rotary evaporatorand cold diethyl ether was added to precipitate the peptide. The peptideprecipitate was collected by centrifuging at 3500 rpm for 5 minutes, thesupernatant was decanted off, and the peptide was washed with coldether. This process was repeated 5×. After the final centrifugationstep, the peptide was dried over vacuum for 60 minutes, dissolved in 25mL deionized water, frozen, and lyophilized to dryness. The dry peptidewas analyzed by MALDI-TOF-MS. Peptide was purified to greater than 95%purity using an acetonitrile/water gradient on a Varian ProStar HPLCsystem equipped with a Grace-Vydac C18 reverse phase column. Forphosphonate-Q11 synthesis, C-SGSG-Q11 was first dissolved in DI H₂O.DMSO containing maleimido-EG6-ethyl-p-nitrophenyl phosphonate was thenadded to the aqueous C-SGSG-Q11 solution and the reaction was allowed toproceed at 37° C. for 2 hours. At the end of the reaction, the solutionwas loaded directly onto a reverse-phase semi-prep scale C18 column andpurified with a linear gradient of 75% can in DI H₂O+0.1%Trifluoroacetic acid over 1 hour. Fractions were lyophilized to drynessand analyzed with MALDI-TOF-MS.

Transmission Electron Microscopy—A 1 mM peptide solution was allowed tofibrillize in PBS for 4 h at room temperature or overnight at 4° C., atwhich point it was diluted to 0.25 mM and pipetted onto carbon-coated200 mesh lacey grids (Electron Microscopy Sciences). The grids werestained with 1% uranyl acetate for 2 minutes, and imaged with a FEITecnai F30 transmission electron microscope.

Reacting phosphonate-Q11 fibrils with cutinase-GFP—Dry Q11 andphosphonate-Q11 were dissolved in deionized water at a 99:1 molar ratioto achieve a final concentration of 10 mM. 1× phosphate-buffered salinewas added to the aqueous peptide solution to achieve a finalconcentration of 1.1 mM. A buffered solution containing 150, 90, 45, 18,or 9 μm GFP-cutinase was added to the buffered peptide solution toachieve a final peptide concentration of 1 mM and a final proteinconcentration of 15, 9, 4.5, 1.8, or 0.9 μM. The reaction betweencutinase-GFP and phosphonate-Q11 was allowed to proceed at roomtemperature for at least 10 hours. The extent of reaction betweenphosphonate-Q11 and cutinase-GFP was monitored by measuring theabsorbance of the solution at 403 nm at specified time intervals. At thereaction end-point, unreacted cutinase-GFP was removed from thecutinase-GFP-modified fibrils by centrifuging the reaction solution at13000 rpm for 5 min, removing 70% of the supernatant with a pipet,replacing the removed volume of supernatant with fresh 1× PBS, andresuspending the fibril pellet by pipetting. This process was repeated 5times to ensure removal of unreacted protein. After removing unreatedcutinase-GFP, the concentration of cutinase-GFP conjugated tophosphonate-Q11 fibrils was quantified by measuring fluorescenceintensity using a 395/509 (excitation/emission) filter set and comparingthe fluorescence intensity readings to those collected from a standardcurve of GFP fluorescence.

Immunization protocols—1 mM Q11:phosphonate-Q11 (99:1 molar ratio) and4.5 cutinase-GFP were mixed under sterile conditions and allowed toreact overnight at room temperature. Unreacted cutinase-GFP was removedfrom the fibrils and the concentration of cutinase-GFP conjugated tofibrils was determined using the methods outlined above. For thisparticular study, the concentration of cutinase-GFP conjugated tofibrils was determined to be 3.58 μm. At day 0, C57BL/6 mice (n=5) wereimmunized by subcutaneously injecting 50 μL of the cutinase-GFPconjugated fibril solution into each of two flanks (100 μL totalinjection volume). At this same time point, separate groups of C57BL/6mice (n=5) were also immunized by subcutaneously injecting 50 μL ofsolution containing 7.16 μm cutinase-GFP emulsified with completeFreund's adjuvant at a 50/50 (v/v) ratio (positive control) or 3.58 μmcutinase-GFP in 1× PBS (negative control) into each of two flanks (100μL total injection volume). At day 31, mice were boosted bysubcutaneously injecting 25 μL, of the cutinase-GFP conjugated fibrilsolution, 7.16 μm cutinase-GFP emulsified with incomplete Freund'sadjuvant at a 50/50 (v/v) ratio (positive control) or 3.58 μmcutinase-GFP in 1× PBS (negative control) into each of two flanks (50 μLtotal injection volume). Blood was drawn from each mouse weekly, serumwas separated from the blood by centrifugation, and total serum IgG wasquantified by ELISA. For ELISAs, microtiter plates were coated overnightat 4° C. with 100 μL of either cutinase-GFP-phosphonate-Q11 fibrils orcutinase-GFP. Control wells were incubated with 100 μL of 1× PBS. Afterovernight coating, plates were washed 3× with 1× PBS containing 0.05%(v/v) Tween-20 (PBST). Plates were blocked with 0.1% bovine serumalbumin in PBST (BSA-PBST) for 2 hours at room temperature. Serum wasserially diluted 1:100 to 1:100000000 in BSA-PBST and 100 μL of eachserum dilution was added to antigen-coated wells or control wellsimmediately after BSA blocking. Serum antibodies were allowed to bind tothe plates for 60 minutes at room temperature, at which point plateswere washed 5× with PBST. 100 μL of horseradish peroxidase-conjugatedgoat anti-mouse IgG (1:5000 dilution) in BSA-PBST was added to thewells. After a 45 minute incubation at room temperature, wells werewashed 5× with PBST. 100 μL 1× TMB substrate was then added to the wellsand allowed to react for 5 minutes at room temperature. At thereaction-endpoint, the reaction was quenched by adding 50 μL 1 M H₃PO₄to the wells. 100 μL of each solution was then transferred to a new wellof a microtiter plate and the absorbance of each solution at 450 nm wasmeasured using a plate reader. Absorbance from control wells wassubtracted from absorbance from antigen coated wells and these resultingvalues, as well as their associated mean, were plotted for each timepoint.

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1. An immunogenic composition comprising a peptide fibril coupled to aplurality of antigens.
 2. The composition of claim 1, wherein thepeptide fibril comprises a plurality of self-assembling peptides.
 3. Thecomposition of claim 2, wherein peptide fibril has a length of at least0.5 to 100 μm.
 4. The composition of claim 2, wherein the peptide fibrilhas a molecular weight of at least 10,000 da-7×10⁸ da.
 5. Thecomposition of claim 2, wherein the self-assembling peptide comprises anamino acid sequence of QQKFQFQFEQQ; KFQFQFE; QQRFQFQFEQQ; QQRFQWQFEQQ;FEFEFKFKFEFEFKFK; QQRFEWEFEQQ; QQXFXWXFQQQ (Where X denotes ornithine);FKFEFKFEFKFE; FKFQFKFQFKFQ; AEAKAEAKAEAKAEAK; AEAEAKAKAEAEAKAK;AEAEAEAEAKAKAKAK; RADARADARADARADA; RARADADARARADADA;SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG; EWEXEXEXEX (Where X=V, A ,S, or P);WKXKXKXKXK (Where X=V, A, S, or P); KWKVKVKVKVKVKVK (Where X=V, A, S, orP); LLLLKKKKKKKKLLLL; VKVKVKVKVDPPTKVKVKVKV; VKVKVKVKVDPPTKVKTKVKV;KVKVKVKVKDPPSVKVKVKVK; or VKVKVKVKVDPPSKVKVKVKV; VKVKVKTKVDPPTKVKTKVKV.6. The composition of claim 5, wherein the self-assembling peptide is 6to 40 amino acids in length.
 7. The composition of claim 2, wherein theantigen is covalently coupled to the self-assembling peptide.
 8. Thecomposition of claim 7, wherein the antigen is covalently coupled to aterminus of the self-assembling peptide.
 9. The composition of claim 8,wherein the antigens are covalently coupled to the carboxy terminus ofthe self-assembling peptide.
 10. The composition of claim 1, wherein theantigen are peptides.
 11. The composition of claim 10, wherein thepeptides are 5 to 20 amino acids in length.
 12. The composition of claim10, wherein the antigens are T cell and/or B cell epitopes.
 13. Thecomposition of claim 1, wherein the antigens are polypeptides.
 14. Thecomposition of claim 13, wherein the polypeptides are covalently coupledto the peptide fibril.
 15. The composition of claim 14, wherein thepolypeptides are covalently coupled to the peptide fibril via a cutinasepolypeptide.
 16. A self-assembling antigen comprising an antigen coupledto a fibril-forming peptide.
 17. A method of inducing an immune responsecomprising administering an antigenic fibril, comprising aself-assembling peptide coupled to an antigen, in an amount sufficientto induce an immune response.
 18. A method of treating a subject havingor at risk of developing a microbial infection comprising administeringto the subject an effective amount of a composition of claim 1.